Enzymic assay of tropic acid esters

Enzymic

A

reccvlt

compwatiw

ported

clicmicnl

flOlll~~i

:~lli:~lOiClS

Bccnusc (2 I , it. accnwtl In acicl

and

its

of of

a high

:lcGl

ntleiiinc~

was

of

the

of the

of tropic:

A possible

wquenw

analytica useful

re-

bella-

methods

in the

and by

tropic rf

:wicl

a&l

cit.

in

(NAI)‘) il

to molt

of rcwtioris

and the

girt ii

~;llow~

for

(TAT>)

reported

and, of

a simiial

of 0.04

in Schr~mc~

ntropine

following

cscess

of NADH

(reduced TU wniicarhazide)

1.

I

Tropic

Acid

Tropic

Acid z$+yH-Co,

H + NADH

CHO Acid -c

c.@-CH-C02H I dH0

+ Tropine

+ NAD + Dehy;;D;“ase

b.,&CH-CO,H I CH,OH

OCH,

CHO

NAo’

0-CH-CO,H

(TaDj

hOZH

TAD ~nc;+0CH~

+ NADH

294

CO,H

was this,

the

nicot~intlmide-

Atropinase a. Atropine-

tropic

microbiological

system,

I)r~wncc

became

dehytlrogcnase

tropine

2 molts

the

specificity

routine

!3 1 ha\-cx

prcw~lcr

in

SCHEME

Tropic

analysis

atropinnse

c,rtc~r:\sc-tl(,llytlro~(,~l~l~~ trol)ic

dchytil~ogc~t~st~~tl :tcitl

induction,

obt:dnvtl

Bervncls

to

cli~~ho~phatc

mole

enzymic

of sensitivity

containcvl

ntropinaso. 2 ~~xipl~l

tropic per

inadequacy

determination

method

enzyme

tlcgrw

:~l>o

to 1)~ hydrc~lyzctl

on

pwlwation,

cwzpicx

foun~l

uf

;1n enzym(~

a stlltly

~~zymc~

ot,hcr

preparat,ion By m~wis

the

caters.

tcchniquw,

m:wy

Esters

the

for

~vsatility to wck

with Thv

in(luction nntl

Iccognizv(l

clispl:Iying

esters.

tlemonst~rated

l~iwwdurc~s

rcnsonablc

wlljunction

n~~nilnblc,

Acid

I1 1 . the

aritl

of Tropic

stucly

analytical

of

of tropic

Assay

NADH

NAD+) .

NADH

NA6

To

+ H++

+

analyze

Resozurin (non-fluorescent)

Resorufin (fluorescent) for

tropic

+

acid,

diaphorase H,O

Scheme

I, step

c., and

Scheme

2 were

used

296

\Il(‘llI*:I..

ll.\(‘l\l,lC’L~.

\SI)

liK.\.\Il,X

Freehol~l, New Jcrscy I , activity 45 unit,s’ mg. One unit cqu:~lr I ;~inole oi NSD+ redllcctl pvr niinuk at, 25°C untlvt* the conditions ~pc~c’ifiotl(4). Diaplioras~~ was cliluted to 1 I units,,‘ml with dist,illcd water. D-H!losc:~~cxttlitie-L-l~-~~tt~~~~~~~~s~i,~~~)t~~f~ :lrrcl ~-11 yosc,t/(trt? inc-I)-.I&cawphorsz~Zfonnte were prcpnre(l from atrop’ine by frac+ional crpst,allization. Elyoscycm he sdfnfe, atropine srrlfcltc, and ia-fropic crcid (Nut~ritional

Hiochcmicals Corp., Clcvelnnd, Ohioj-stock solution:: of 2 X IO-’ M each were prepared in distilled water. The buffers employed in at,ropinnse studies wcrc: 0.01 ~11phosphate + 0.1 M KC1 for pH 6 to 7.6; 0.01 X tris (hydrosymct~hyl) aminomethane (Tris) + 0.1111 KC1 for pH 7.6 t)o 8.8; 0.05 111pyrophosphnte for pII 8.8 to 9.4; and 0.01 ~$1glycine + 0.1 df KC1 for pH 9.4 to 10.3. 0.1 111 Tris, pH 8.8 to 9.0, was employecl for the dchydrogenase clct.erminations. dppcrrc~ttcs.All fluorcsccnce mea~urrment~smere made with an AmincoBowman sr)ectrophotofluororllctcl as dtxribcd prcriouslp (4). A Radiomctc~r was employed in pII-stat dctrnuinatioll~. X Gary spcctrophotomctcr xxs usvd to perform the sl~c~ctrol~l~ot~oii~~t~ric clctcrminntions.

Procedure 4: (1) Detemination of At~pi~nse. A solution is prepared consisting of buffer 0.01 X Tris containing (0.1 M KCl, pH S.O), I,hyoscyaminc, 2 x 10-j M, and enzyme (j-20 mgm,/ml of protein). Absorbance is obtained in a Cnry spcctrophotonlcter using a 1.0 cm cell and a sensit,irity range set at 0.1. The at8rolGnaseactivity is determined by monitoring t,hc AOD,‘min at, 219 nm, where t,hc maximum change in absorbance occurs on hydrolysis of atropinc. The method is based on the diffcrcnce spectrum between atropinc and its hydrolysis produck The blank consists of L-hyoscynminc and irlcntical enzyme solution (heatccl at 60°C for 1 mill 1. (2)

I)eferminnfion

of

.dtrq~ine

md

I)-

trntl

r.-El!/osc!ic/tttines.

U&now11

solutions containing atropine nl1(1I)- and L-liyoscynmines, in the 10e7to itlntion of Atropinnse) ex10-j 31 rnngc are assayed as nhove (Ilefem cept that t)he hydrolysis is :rllowccl to proceccl to complet~ionand the total A(?D is me:ts11r(d, employing a 10 cm cell. From suit,able calibration rurvcs. t,hc concentration of the sample is determined. Produrc

13: Determination

of ~ltlopinnse

~IJ

pH-stat

Method.

L-

Hyo+cyaniinc is dissolved in 0.1 M KC1 to gire a concentration of lo-” M; I ml of the substrate solution is put into the thermostatted cell of a Radiomc+clr pH-stat maintained at 2.i”C and brought to the desired pH (‘usually pH 71 with 0.01 N sodium hyclrosidc or 0.01 N hydrochloric acid. Then 0.1 ml of a 0.1 .?f KC1 sohltioll containing atrol)inase (2.0 mg/

10 jnl of ().I .lf lit’1

solution c~olit:Aning 0.1 mtnolc~ of L-hyctscyztminesalt, was h~tl~~olyzed with 10 mg of crude (accu-lO-c~~~lll~‘llol~~ulfoll:~t~~ tcJlltA ~10W&1~) L’kzynx’ coi~taiiiing atropinaac at pH 7.0 for 4 hr at 25°C. X\TObuffer was c~ml~loyetl anal the pH RW held constant by means of a salt n-as similarly pH+tnt. Thc~ r~-l~~os;c~a~~li~~~~-~,-lO-c~ntll~~~~or~~~lfo~~~~te trrat,etl for 20 hr. Both solutions wvw brought to I)H 3.0 with I S HCl. saturated with sodium cliloritle, :md tMr.acted with ether (I)eroxiclc-free) Evnporntion of t,htx ctlicbr in :I stro:nn of dry N, at room teniperat,urcx yicIdec1 rcsicluc>h n-llich worth rc>-cstractccl with 2 ml of dry ether. To obtain L-t,ropi(* kc-1, 4 ml of cyclohcxanc~ was :rtl(lccl and the crystal:: were collcct’ecl: 111.1). 130.5-131 o (lit,. 130”) ; 1a-1i:;x,,.“\- 52.50. To obt;Gll thcx D-tropic’ :Acl, 6 1111 ry~lollcxalu~ \v:is ;Icldell to t.hc et~hw solution, Cva1)orated to 3..5 ml volume, and :~llo~~d to crystnllizc: tn.p. 12%129” ; [ru ( ..:!& 5210.

p11

Km

6 00

:;.f5

x

10-6

S.iI)

5.4 1.7 :JJ.o 4.2

x x x x

10-e 10-s 10-s 10-6

0 .I‘30 9.5x !I .50

occurs up to pH 8.7 but, a large increase occurs between pH 8.7 and 9.7. The pH-activity profilr of atropinnse is given in Table 2. There was no pH optimiurn for the clnzyme activity, but it approarhed a maximum at. pH 10. Tl1es.c~ dct.erminat.ions were obtained at virtual V,,,,, values (2.0 X 10m4111 L-hyoscyaminel, and indicated that enzyme activity is ~~roh:tbly clqx~ntlt~nt on a group in the enzyme with a pk’, of about 6.0.

8rhs(rate

l-Hyoscyamille u-IIyoscyamiue uL-Hyoscyamine (ntropine, Scopolamine DL-~I et hyl tropate Diethyl phenylmaloxttc Ethyl phenylacetate Ethyl cinnamate Ethyl phenylglyosal;lt e Ethyl lactnk I)L-Tropic acid hydrnzitlc Benzyl chloroaceI:rie Methyl benzilatc 2,6-L~ic~hloroindoJ)hel~~l:~~~~~it,e

J 00 2 50 6; 14,’ 11” 0” I I’L 0” (P’ I I” I I” 0” 0

Lkkrminntions w-ere rrm on crrlde atropiuasr, JIII 7.4. n pH-stat met,hotl. Othw detrrmiw~tions U-ere done spect ruphotomet

rically.

The & values for the dehydrogenase were determined for D- and Ltropic acids using Procedure D. At pH 8.8, 0.05 M pyrophosphate buffer, and 1.86 X 10ebM N,4D’ the Ki’, value for o-kopic acid was 1.4 X 10 4 while, for L-tropic acid, it was 4.8 x 10-j. I’,,,,, for the n-isomer was 1.43 x IO-” OD/min while for t’he L-isomer it was 1.26 X lo-” OD,/min. The effect of the greater affinity of the L-isomer for the dehydrogrnsse is manifested in greater activity at the lower concentrations. A study was carried out to determine the substrate specificity for the dehydrogenase. The react,ion of tropic acid dehydrogenasc wit.h various substrates ie shown in Table 4. The enzyme is highly specific for tropic acid. Substrate

TBBLE of Tropic

Specificity

4 Acid

Substrate -

Dehydrogenasea Relative

activity

-I_-__-___

n-Tropic Acid L-Tropic Acid L-Hyoscyamine n-Hyoscyamine Methyl tropate Tropic acid hydrazide Benzylamine Ethanol Tropine Benzilic acid a Det,erminstion buffer.

100
0.1 0 (I 0 0 0 0 was made

on purified

dehydrogenase,

pH

5.8, 0.05 M pyrophosphate

Attempts were made to substitute NADP’ (triphosphopyridine dinucleot,ide) for NAD+ using the dehydrogenase and L-hyoscyamine. iYo activity leas obtained. Determination of L-Hyoscyanzine. Using Procedure A, L-hyoscyamine wa.s det,erminetl and the results are given in Table 5. L-Hyoscyamine may Delermination L-Hyoscyamine .~-

concn. 0 (blanki 1.1’ 2.24 .i.GO 11.2 13.4

TABLE 5 of kHyoscyamine X lo7 ilf

[Procedure

A]

Total

OD

0.0017 0.0035 0. 0055 0.0097 0.0183 0.0216

he determined in the range of lC7 to 10e4M with a standard deviation of 24 .x 10-S nr. Employing the coupled enzyme syskm, and monitoring the over-all reaction with NAD+/resazurin,kliaphorase, the concentrations of atropine, L-hyoscyamine, and m-tropic acid were measured with a fair degree of accuracy in the 7 x IO-” to 1 X lo-” M range. The rate method was used because it was more rapid than t,hc total change method. The order of eensitiritp was L-hposcyaminc > rs-t#ropic acid > atropinc (Fig. 1). This

.2

0

FIG.

1. Determination

.4

of tropic

.6

.8

CONC

X

arid

IO

and

1.2

I .o -6

M/L

its esters

I,-

fluorcsecnw

would bc expected on the basis that L-hyoxyamine is more rapidly hydrolyzed than atropinc since the ntropinnse at.tacks the L-isomer at a grea,tcr rate t,han the n-isomer and atropine is a nL-mistllre. Further, with respect to x-tropic arid, the L-isomer is more r:ipiclly oxidized in t,he lower concentration ranges t,han the n-isomer because of the more favorablr Ii, value.

REFERENCES I. R.. AkHM~D. 55, 16916 (1961). GUILBAULT, G. G.. ilwzl.

1. F4HMY, 2.

Z.

F.,

Chem..

AND

EID,

S.

Ann.. Rev.

A.,

J.

Chem.

40, 459R

TJAR,

(1968).

3, 229 (1960);

C. A.